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Cysteine transport through excitatory amino acid transporter 3 (EAAT3)

Watts, SD and Torres-Salazar, D and Divito, CB and Amara, SG (2014) Cysteine transport through excitatory amino acid transporter 3 (EAAT3). PLoS ONE, 9 (10).

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Abstract

Excitatory amino acid transporters (EAATs) limit glutamatergic signaling and maintain extracellular glutamate concentrations below neurotoxic levels. Of the five known EAAT isoforms (EAATs 1-5), only the neuronal isoform, EAAT3 (EAAC1), can efficiently transport the uncharged amino acid L-cysteine. EAAT3-mediated cysteine transport has been proposed to be a primary mechanism used by neurons to obtain cysteine for the synthesis of glutathione, a key molecule in preventing oxidative stress and neuronal toxicity. The molecular mechanisms underlying the selective transport of cysteine by EAAT3 have not been elucidated. Here we propose that the transport of cysteine through EAAT3 requires formation of the thiolate form of cysteine in the binding site. Using Xenopus oocytes and HEK293 cells expressing EAAT2 and EAAT3, we assessed the transport kinetics of different substrates and measured transporter-associated currents electrophysiologically. Our results show that L-selenocysteine, a cysteine analog that forms a negatively-charged selenolate ion at physiological pH, is efficiently transported by EAATs 1-3 and has a much higher apparent affinity for transport when compared to cysteine. Using a membrane tethered GFP variant to monitor intracellular pH changes associated with transport activity, we observed that transport of either L-glutamate or L-selenocysteine by EAAT3 decreased intracellular pH, whereas transport of cysteine resulted in cytoplasmic alkalinization. No change in pH was observed when cysteine was applied to cells expressing EAAT2, which displays negligible transport of cysteine. Under conditions that favor release of intracellular substrates through EAAT3 we observed release of labeled intracellular glutamate but did not detect cysteine release. Our results support a model whereby cysteine transport through EAAT3 is facilitated through cysteine de-protonation and that once inside, the thiolate is rapidly re-protonated. Moreover, these findings suggest that cysteine transport is predominantly unidirectional and that reverse transport does not contribute to depletion of intracellular cysteine pools.


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Details

Item Type: Article
Status: Published
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Watts, SDsdwatts@pitt.eduSDWATTS
Torres-Salazar, D
Divito, CB
Amara, SG
Contributors:
ContributionContributors NameEmailPitt UsernameORCID
EditorBoudko, DmitriUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Centers: Other Centers, Institutes, Offices, or Units > Center for Neuroscience
Date: 2 October 2014
Date Type: Publication
Journal or Publication Title: PLoS ONE
Volume: 9
Number: 10
DOI or Unique Handle: 10.1371/journal.pone.0109245
Schools and Programs: School of Medicine > Neurobiology
Refereed: Yes
Other ID: NLM PMC4183567
PubMed Central ID: PMC4183567
PubMed ID: 25275463
Date Deposited: 12 May 2015 18:03
Last Modified: 21 Jan 2019 20:55
URI: http://d-scholarship.pitt.edu/id/eprint/24025

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